In this project the structures and biosynthesis of glycoprotein and glycolipid antigens of malignant melanoma, originally detected by human serum antibodies and mouse monoclonal antibodies, will be analyzed. We have previously shown that the sera of some patients contain antibodies reacting only with autologous tumor cells. One of these antibodies (FD) is immunoprecipitating and detects a glycoprotein of 90,000 kD. The glycoprotein is also present on other cells but these forms lack the determinant detected by serum FD. This glycoprotein (gp90) will be purified and its biochemical characteristics, including partial amino acid sequence and glycosylation features, will be analyzed. Attempts will be made to clone the gene coding for gp90 using lambda gt11 expression libraries, oligonucleotide probes, and transfection approaches. Comparison of protein and nucleotide data for gp90 from the autologous melanoma with that from other cell types should demonstrate the basis for the FD restricted epitope. The aim is to understand the significance of restricted antigens such as FD and the immune response to them in patients. We will also continue our studies of gangliosides characteristic of malignant melanoma, particularly GD3. A key step resulting in the expression of this ganglioside is the activity of the enzyme (GD synthetase; CMP-N- acetylneuraminic acid:GM3 sialyltransferase) that converts GM3 to GD3 since normal melanocytes have extremely low levels of GD3. The enzymatic properties of this enzyme including detergent requirements, pH optimum, ion requirements, Km for acceptor and donor, etc., will be studied. Also, the activity of the transferase in different cell types will be assayed to determine whether GD3 levels are controlled mainly by enzyme levels. The sialyltransferase will be purified using biochemical and affinity chromatography methods and its biochemical characteristics, including partial amino acid sequence and glycosylation, will be analyzed. Monoclonal antibodies to the purified enzyme will be produced and used to further analyze its properties and distribution. Experiments will also be initiated to clone the gene encoding GD3 synthetase using transfection and other strategies. Overall goals of this project are to understand the control of restricted determinant expression and of characteristic glycolipid synthesis and also to provide reagents for the immunodiagnosis and therapy of melanoma.